Two-photon calcium imaging (2PCI) allows the simultaneous visualization and physiological characterization of hundreds of neurons within a small patch of cortex with single neuron resolution. With the advent of genetically-encoded Ca2+ indicators (GECIs), it is now possible to identify and track neurons for extended periods, up to a month or longer. This opens up new opportunities for the thorough characterization of neurons and for longitudinal studies that examine the neural bases of learning. While awake 2PCI is a well-established technique in smaller animals, its development is still in its infancy in the primate. Our goal is to successfully implement 2PCI in the awake behaving macaque and establish it as a powerful tool in the arsenal of primate systems neuroscientist. This proposal addresses the two major challenges to successful 2-photon imaging in the awake macaque. First, an imaging chamber with a clear, transparent window and a suitable interface to the 2P microscope objective must be implanted and maintained free of tissue growth for a prolonged period in the awake animal. Second, a protocol for the reliable expression of a GECI must be developed for the macaque. We will work independently on each of these challenges in Aims 1 and 2 and will combine the resulting technologies in Aim 3. In Aim 1, we will implant a custom-designed low profile chamber, perform a craniotomy and durotomy in a bloodless surgery and implant an artificial dura. We will then refine the technique to maintain the chamber over months by removing the neomembrane regrowth in a delicate bloodless procedure. We will express GFP within the chamber and assess the quality of 2P imaging over the course of months. We will also implement hardware and software strategies for image stabilization and alignment, both within session to correct for motion artifacts and to identify matched neurons across sessions. In Aim 2, we will identify the appropriate AAV capsid serotypes, promotors, and injection procedures to express GCaMP6 by performing injections with a variety of parameters and assessing expression in postmortem tissue. We will also assess the stability of the GCaMP signal over time, its signal-to-noise ratio, its linearity, its toxicity to cells and the toxicity f the laser to the cells in anesthetized animals. Finally, in Aim 3 we will conduct 2P experiments in the awake animal with the GECI expressed using the procedures refined in Aim 2. We will quantify the tuning of V1 neurons for basic physiological parameters (orientation, spatial frequency, etc.) across weeks and months to determine whether the signals are sufficiently stable to allow long-term studies of neurons. Our experiments will provide the first detailed evaluation of whether 2P imaging in the awake macaque can serve as a powerful tool for longitudinal studies and visualization of neurons, and our results will provide a recipe for implementation of this technique that can be easily transplanted to other labs. Ultimately, this technique can help us understand how networks of neurons underlie learning and complex behavior, and it can aid in devising strategies to alleviate brain disorders in which these capacities are impaired.